EGFR- and Integrin αVβ3-Targeting Peptides as Potential Radiometal-Labeled Radiopharmaceuticals for Cancer Theranostics

The burgeoning field of cancer theranostics has witnessed advancements through the development of targeted molecular agents, particularly peptides. These agents exploit the overexpression or mutations of specific receptors, such as the Epidermal Growth Factor receptor (EGFR) and αVβ3 integrin, which are pivotal in tumor growth, angiogenesis, and metastasis. Despite the extensive research into and promising outcomes associated with antibody-based therapies, peptides offer a compelling alternative due to their smaller size, ease of modification, and rapid bioavailability, factors which potentially enhance tumor penetration and reduce systemic toxicity. However, the application of peptides in clinical settings has challenges. Their lower binding affinity and rapid clearance from the bloodstream compared to antibodies often limit their therapeutic efficacy and diagnostic accuracy. This overview sets the stage for a comprehensive review of the current research landscape as it relates to EGFR- and integrin αVβ3-targeting peptides. We aim to delve into their synthesis, radiolabeling techniques, and preclinical and clinical evaluations, highlighting their potential and limitations in cancer theranostics. This review not only synthesizes the extant literature to outline the advancements in peptide-based agents targeting EGFR and integrin αVβ3 but also identifies critical gaps that could inform future research directions. By addressing these gaps, we contribute to the broader discourse on enhancing the diagnostic precision and therapeutic outcomes of cancer treatments.


Introduction
The area of cancer theranostics has seen remarkable progress, with the development of several targeting agents, particularly peptides.These molecules exploit the upregulation of specific receptors, such as the Epidermal Growth Factor receptor (EGFR) and integrin α V β 3 .These receptors recognize specific amino acid sequences like tyrosine kinase domaintargeting peptides and RGD (Arg-Gly-Asp) derivatives, respectively, making them prime targets for therapeutic and diagnostic applications [1,2].
Peptides are particularly notable in this context due to their low molecular weight, generally consisting of up to 50 amino acid residues.This attribute allows for enhanced penetration into tumor tissues and swift clearance from the bloodstream and non-targeted tissues, which improves imaging accuracy and reduces treatment-related toxicity.Additionally, peptides are characterized by low antigenicity, and they can undergo chemical modifications during the radiolabeling process without losing activity, further enhancing their suitability for theranostic applications [3,4].Over the past decades, the identification and utilization of peptides that target EGFR and integrin α V β 3 have significantly increased.
In this comprehensive review, we provide a thorough summary of the current literature concerning the utilization of radiolabeled EGFR-and integrin α V β 3 -targeting peptides in cancer imaging and peptide receptor radionuclide therapy (PRRT).We discuss the various EGFR-and integrin α V β 3 -targeting peptides developed to date, emphasizing their efficacy as targeting ligands and outlining their advantages and disadvantages when compared to alternative targeting agents.Our discussion extends to their applications in both preclinical and clinical studies, whether labeled with common imaging radionuclides for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) or with therapeutic radionuclides for cancer radionuclide therapy.Lastly, we assess the challenges associated with translating these radiolabeled peptides to clinical use and contemplate the prospects for future research in this field.

EGFR and Integrin α V β 3 as Cancer Targets
The EGFR, also called ERBB-1 (Erythroblastic Leukemia Viral Oncogene Homologue-1) or HER-1 (Human Epidermal Growth Factor Receptor-1), is a 170 kDa single-chain transmembrane glycoprotein belonging to the ERBB family of receptor tyrosine kinases.EGFR presents three domains: (a) a hydrophobic transmembrane domain, which is associated with dimerization interaction among receptors; (b) an intracellular tyrosine kinase domain responsible for substrate phosphorylation; and (c) an extracellular EGFR-binding domain (sEGFR), which binds ligands which stimulate EGFR [5,6].The sEGFR consists of four domains (I, II, III and IV), and exhibits two conformations: an open conformation when bound to a ligand and a closed, auto-inhibited conformation [1].Ligand binding induces conformational changes, exposing a dimerization arm that facilitates the formation of homodimers and heterodimers with other ERBB family members.This dimerization activates the kinase domain, which consists of two lobes, with a cleft between them containing four critical structural sites: (1) the catalytic region, (2) the hinge site, (3) the activation loop, and (4) the kinase specificity pocket [7][8][9][10].
The aberrant activation of those EGFR signaling pathways, due to factors like cellsurface overexpression, autocrine activation, and mutations in the EGFR gene, is observed in various cancers, including lung, head and neck, cervical, colorectal, and brain cancers [11].Among the various EGFR mutations, the most prevalent is EGFRvIII, an oncogene formed by a large extracellular deletion [12].
While EGFR itself is a critical target in cancer therapy due to its overexpression and mutations in various tumors, it is essential to recognize that the downstream signaling pathways activated by EGFR are the primary drivers of cancer progression.EGFR activation triggers two key signaling cascades in cancer: the PI3K/AKT/mTOR and MAPK pathways.The PI3K/AKT/mTOR pathway regulates cell growth, proliferation, and survival [13,14], while the MAPK pathway controls gene expression and cell cycle progression [11,15].Both pathways are frequently dysregulated in cancers, contributing to uncontrolled growth and therapy resistance.NF-κβ [16] and GTPase path signaling are also activated [17].The result of these intracellular pathways is the increased transcription of genes responsible for cell growth, survival, and/or migration [18].Significant crosstalk between these pathways can lead to compensatory mechanisms, making single-pathway inhibition often ineffective.Understanding these interactions is crucial for developing combination therapies that target multiple pathways simultaneously to overcome resistance and improve cancer treatment outcomes.
On the other hand, integrins are a family of transmembrane cell surface receptors that mediate cell-matrix and cell-cell interactions [19].These heterodimeric glycoproteins consist of noncovalently connected α and β subunits.In mammals, 18 α and 8 β subunits combine to form at least 24 distinct integrin heterodimers [20,21].Each integrin subunit has a large extracellular domain (750-1000 residues), a single transmembrane domain, and a short cytoplasmatic domain [22,23].Integrins bind to specific endogenous ligands, including cell surface counter-receptors, soluble ligands, and extracellular matrix (ECM) proteins [24].Eight integrin heterodimers can recognize RGD-containing peptides [25].Ligand binding triggers interactions between the cytoplasmatic domain, intracellular proteins, and cytoskeletal filaments, initiating signaling pathways through the Src kinases, which are activated by FAK phosphorylation [26].This signaling controls key cellular functions, including adhesion, migration, proliferation, differentiation, and survival, that are critical for tissue homeostasis, development, and repair [27].Integrin dysregulation is involved in the pathogenesis of several diseases characterized by altered angiogenesis, inflammation, or infection [28].
Integrin α V β 3 , also known as the vitronectin receptor, is of particular interest in cancer research.While α V β 3 is expressed at low levels or is undetectable in most healthy adult epithelia, it is overexpressed in various tumor cells (breast, lung, glioblastoma, prostate, and melanoma), the tumor-associated vasculature, and invasive tumor fronts [23].Interactions between α V β 3 and its ECM ligands activate intracellular signaling pathways like PI3K/Akt and ERK/MAPK, promoting cancer progression.The engagement of α V β 3 with the ECM also facilitates crosstalk with growth factor receptors and proteases to further enhance tumor cell survival and motility [29].Notably, α V β 3 expression is associated with the acquisition of a cancer stem cell phenotype, conferring tumors with enhanced initiation and therapy resistance capabilities [30].
Therefore, targeting EGFR and/or integrin α V β 3 with specific inhibitors or antibodies/small molecules can disrupt these signaling pathways, inhibiting tumor growth and spread.

EGFR-Targeting Peptides
Several EGFR-targeting peptides have been identified and developed as potential diagnostic and therapeutic agents.EGFR peptides can be obtained from experimental synthesis, phage display libraries, computer-aided design, and natural sources.The most widely explored peptide is GE11 [31][32][33][34][35][36][37][38].GE11 is a dodecapeptide that is identified through phage display screening.It exhibits high affinity and selectivity for EGFR.It binds to EGFR with a dissociation constant (Kd) of 22 nM, which is lower than that of the natural ligand EGF (Kd = 2 nM) but significantly higher than seen for non-specific binding to proteins like bovine serum albumin.This lower affinity compared to EGF is attributed to GE11's smaller size and its binding to only one EGFR region, in contrast to EGF's interaction with multiple domains [18].De Paiva et al. [39] identified the binding site between GE11 and sEGFR using molecular dynamics and molecular docking simulation.According to these researchers, GE11 acts as an inhibitor.The optimal conformation of GE11 and sEGFR occurs at domains II and IV, which may block the exposure of the dimerization arm and prevent dimer formation [1,40].Importantly, while GE11 binds to EGFR with high specificity, it does not exhibit mitogenic activity.This characteristic makes it an attractive option for targeted drug delivery without stimulating tumor growth.Recent studies have shown that GE11 can enhance nanoparticle endocytosis through an alternative EGFR-dependent, actin-driven pathway [18].This mechanism allows for the maintenance of EGFR levels on the cell surface after GE11 binding, potentially enabling prolonged receptivity to GE11-conjugated therapeutics.Other peptides like EBP (CMYIEALDKYAC) and D4 (LARLLT) have also shown promise in EGFR targeting.EBP was experimentally synthesized and demonstrated high affinity for EGFR, while D4 was developed through computer-aided design approaches [1].Recent research by Tripathi et al. (2024) [16] demonstrated the anticancer potential of short peptides, derived from the conserved regions of the MIEN1 protein.Their study highlighted that a six-amino-acid peptide, LA3IK, effectively inhibited EGF-mediated NF-kB nuclear translocation in breast cancer cells.This finding underscores the therapeutic promise of targeting the MIEN1 signaling pathway to impede cancer progression.
In addition to targeting wild-type EGFR, there has been significant progress in developing peptides that are specific to EGFR mutations, such as EGFRvIII, which is commonly associated with aggressive cancers like glioblastoma.EGFRvIII, characterized by the deletion of exons 2-7, leads to a constitutively active receptor that drives tumorigenesis.Recent studies have utilized phage display technology to identify cyclic peptides that selectively bind to EGFRvIII.For instance, novel cyclic peptides P6 and P9 have shown high specificity for EGFRvIII-expressing cells, enhancing targeted drug delivery and cytotoxicity in non-small-cell lung cancer (NSCLC) and glioblastoma models [41].These advancements underscore the potential of peptide-based therapies to effectively target specific EGFR mutations, offering new avenues for precision oncology.
These peptides, along with others listed in Table 1, represent a diverse array of EGFRtargeting strategies, each with unique binding properties and potential applications in cancer diagnostics and therapeutics.

c(RGDf[NMe]V (cilenglitide) and RGDechi
Cilenglitide acts as a selective α V β 3 antagonist.Preclinical studies have demonstrated its antiangiogenic and antitumor effects in various cancer models [45].While cilengitide was well tolerated in phase I/II clinical trials [46,47], it failed to show efficacy in phase III studies [48], likely due to the complexity and plasticity of integrin signaling networks.RGDechi is a designed α V β 3 antagonist based on cilenglitide structures combined with echistatin C-terminal tails [57]. ) is a capped pentapeptide synthesized from the fibronectin-PHSRN sequence and it can be used alone or along with radiotherapy and chemotherapy to prevent metastasis and tumor development [49][50][51].In phase I of a clinical study, ATN-161 was used for aggressive solid tumors and demonstrated good toleration and safety [53].

RWrNK and RWrNM
RWrNK and RWrNM are linear peptides that contains an unnatural d-arginine (r).They present great water solubility and the ability to pass through the blood-brain tumor barrier.They have been investigated for glioblastoma diagnosis [58,59].

Radiolabeled Peptides as Valuable Tools for Imaging and the Treatment of Cancer
The process of radiolabeling peptides involves attaching radioactive isotopes to peptides, which can then be detected using imaging techniques to provide real-time, noninvasive insights into the molecular environment of tumors.This capability not only aids in the accurate diagnosis and staging of cancer but also facilitates the monitoring of treatment responses and the detection of metastases.Furthermore, when these radiolabeled peptides are designed to carry therapeutic radionuclides, they serve a dual function by also providing targeted radionuclide therapy, delivering cytotoxic radiation directly to tumor cells and thereby reducing the tumor burden while sparing normal tissues [60].

Strategies for Radiolabeling Peptides
Peptide radiolabeling employs two primary methods: direct and indirect labeling (Figure 1).Cilenglitide acts as a selective αVβ3 antagonist.Preclinical studies have demonstrated its antiangiogenic and antitumor effects in various cancer models [45].While cilengitide was well tolerated in phase I/II clinical trials [46,47], it failed to show efficacy in phase III studies [48], likely due to the complexity and plasticity of integrin signaling networks.RGDechi is a designed αVβ3 antagonist based on cilenglitide structures combined with echistatin Cterminal tails [57].
Non-RGD peptides ATN-161 ATN-161 (Ac-PHSCN-NH2) is a capped pentapeptide synthesized from the fibronectin-PHSRN sequence and it can be used alone or along with radiotherapy and chemotherapy to prevent metastasis and tumor development [49][50][51].In phase I of a clinical study, ATN-161 was used for aggressive solid tumors and demonstrated good toleration and safety [53].

RWrNK and RWrNM
RWrNK and RWrNM are linear peptides that contains an unnatural d-arginine (r).They present great water solubility and the ability to pass through the blood-brain tumor barrier.They have been investigated for glioblastoma diagnosis [58,59].

Radiolabeled Peptides as Valuable Tools for Imaging and the Treatment of Cancer
The process of radiolabeling peptides involves a aching radioactive isotopes to peptides, which can then be detected using imaging techniques to provide real-time, noninvasive insights into the molecular environment of tumors.This capability not only aids in the accurate diagnosis and staging of cancer but also facilitates the monitoring of treatment responses and the detection of metastases.Furthermore, when these radiolabeled peptides are designed to carry therapeutic radionuclides, they serve a dual function by also providing targeted radionuclide therapy, delivering cytotoxic radiation directly to tumor cells and thereby reducing the tumor burden while sparing normal tissues [60].

Strategies for Radiolabeling Peptides
Peptide radiolabeling employs two primary methods: direct and indirect labeling (Figure 1).

Direct Labeling
The radioisotope is covalently attached to the peptide.This is commonly performed with radioiodines like iodine-125 ( 125 I, t 1/2 = 59.4 days; Eγ = 35 keV) and iodine-131 ( 131 I, t 1/2 = 8 days; 90% β − = 606 keV) via the electrophilic radioiodination of the tyrosine side chain aromatic ring.Oxidizing agents such as Chloramine T or Iodo-Gen ® are used to convert iodide into an electrophilic iodate that is substituted onto the tyrosine's aromatic ring at room temperature [61].This procedure offers the advantage of not modifying the amino acid sequence.Examples of direct iodination include 125 I-labeling of GE11 [31] and 131 I labeling of GRGDYV [62].
Direct 99m Tc-(t 1/2 = 6 h; Eγ = 140 keV) labeling is also performed for peptides with disulfide bonds or via the formation of a [ 99m Tc(CO) 3 ] + complex that binds the histidine side chain imidazole ring.This two-step approach first makes the [ 99m Tc(H 2 O) 3 (CO) 3 ] + core, which then labels the histidine to form a stable complex, as demonstrated for GRGDHV [62].The study of Baishya and coworkers evaluated two [ 99m Tc(CO) 3 ] + -labeled tetrapeptides and one [ 99m Tc(CO) 3 ] + -labeled hexapeptide by changing the amino acid sequence of the RGD motif for potential use as tumor-targeting radiopharmaceuticals [63].Comparative in vivo studies of [ 99m Tc(CO) 3 ] + -labeled PEGylated and non-PEGylated cRGDfK demonstrated that the addition of a PEG 7 unit increased the melanoma tumor uptake and slowed the clearance from other organs, decreasing target-to-background ratios [64].However, 99m Tc peptide labeling more often uses indirect methods.

Indirect Labeling
The bifunctional chelator or prosthetic group is attached to the peptide, which then complexes with the radiometal.Typically, the chelator is coupled to the peptide first to simplify the radiosynthesis.Linkers can also be added between the chelator and peptide [65].
Over 80% of the radiopharmaceuticals utilized for SPECT imaging rely on 99m Tclabeled compounds due to their favorable nuclear properties and widespread availability through 99 Mo/ 99m Tc generators at a low cost.With a half-life of 6 h, 99m Tc allows radiopharmacists ample time for radiosynthesis, while still permitting physicians to obtain clinically pertinent images.Among the chelating agents employed for the labeling of 99m Tc compounds, HYNIC (6-hydrazinonicotinic acid) is the most extensively utilized [33,76,78].
Other studies cast doubt on GE11's EGFR affinity [36,82].Striese et al. [36,82] and Judmann et al. [36,82] attributed this lack of targeting efficacy to GE11's high hydrophobicity, which may cause peptide aggregation and limit its interactions with EGFR.They also proposed that the cell uptake reported in other GE11 studies may be facilitated by highly hydrophilic linkers or constructs with multiple peptide copies, suggesting that developing small-molecule GE11-based radioligands may not be a promising approach to obtaining alternatives to GE11.Few attempts have been made in terms of other peptide scaffolds [50,[76][77][78]83].Table 7 summarizes all the collected preclinical data.111 In [79], 64 Cu [36,81,132], 99m Tc [33,34,37], and 68 Ga [82, [133][134][135].However, its EGFR-targeting efficacy is controversial. Som studies reported good binding affinity and tumor uptake in cell lines and murine models [31][32][33][34]37]. Figure 2   Other studies cast doubt on GE11's EGFR affinity [36,82].Striese et al. [36,82] and Judmann et al. [36,82] a ributed this lack of targeting efficacy to GE11's high hydrophobicity, which may cause peptide aggregation and limit its interactions with EGFR. Thy also proposed that the cell uptake reported in other GE11 studies may be facilitated by highly hydrophilic linkers or constructs with multiple peptide copies, suggesting that developing small-molecule GE11-based radioligands may not be a promising approach to obtaining alternatives to GE11.Few a empts have been made in terms of other peptide scaffolds [50,[76][77][78]83].Table 7 summarizes all the collected preclinical data.   : not available.

Use of EGFR-Targeting Peptides for Therapeutic Purposes
No studies have yet been conducted that utilize radiotherapeutic isotopes for the treatment of conditions via EGFR-targeting peptides.This gap in the research might be attributed to the controversial efficacy of EGFR targeting itself.This inconsistency in the efficacy of EGFR targeting could potentially prevent the development of therapeutic applications involving radiotherapeutic isotopes, as the foundational premise of specific and effective EGFR targeting remains under debate [36,82].

Integrin α V β 3 -Targeting Peptides
The development of radiotracers for imaging purposes employs various strategies [136].The multimerization of the cRGD scaffold is an approach that leverages the polyvalence effect to increase the binding affinity to integrin α V β 3 .This method suggests that radiotracers derived from multimeric peptides exhibit higher tumor uptake and better tumor/background ratios than their monomeric counterparts [104,137,138].However, this can also lead to increased non-specific uptake in non-target tissues [52].Guo et al. [139] showed that the 18 F-labeled RGD dimer ([ 18 F]F-FP-PRGD2) had a greater binding affinity than the monomer ([ 18 F]F-FP-RGD) in mice bearing MDA-MB-435 tumor xenografts (difference in %ID/g uptake: RGD2/RGD~1.5, p = 0.0045).Chen et al. [68] also demonstrated the higher tumor uptake of [ 18 F]F-FPRGD2 than [ 18 F]F-FPRGD at all time points in a glioblastoma xenograft mouse model.The dimeric tracer showed predominantly renal excretion, while the monomeric tracer was excreted primarily through the biliary route, resulting in higher tumor/background ratios [69].
Pegylation has been shown to extend the radiotracer's circulation time and modulate its clearance, without affecting rapid elimination from the liver and kidneys [25,67].Glycosylation, involving the conjugation of a sugar amino acid to the peptide structure, also enhances hydrophilicity and blood circulation time.One notable example of a glycosylated radiotracer is [ 18 F]F-Galacto-RGD, which demonstrates significant tumor uptake, fast blood clearance, and predominantly renal elimination [25,66,140].
Use of Integrin α V β 3 -Targeting Peptides for Therapeutic Purposes The radioisotopes that are mainly in use are the β − emitters, like Lutetium-177 ( 177 Lu) and Yttrium-90 ( 90 Y) (Table 10) [125][126][127][128][129][130][131]. 177Lu emits both β − particles and γ rays, which allows, respectively, radionuclide therapy and SPECT imaging to confirm the distribution and localization of the therapeutic agent. 177Lu has a half-life of 6.7 d and a tissue penetration range of about 2 mm, making it suitable for treating small-to medium-sized tumors.Conversely, 90 Y is a pure β − emitter with a higher energy and longer range (approximately 11 mm) than 177 Lu.These characteristics make it effective in treating larger tumors.However, its shorter half-life of 2.7 d requires more precise timing in clinical applications [129,144].The efficacy of 177 Lu-and 90 Y-labeled integrin α V β 3 -targeting peptides in animal models has shown promising results.However, some challenges need to be addressed to enhance the efficacy and safety of these therapies: the heterogeneity of tumor expression, the development of resistance, and radiation toxicity (this can affect surrounding healthy tissues) [145].Further research is needed to explore the use of other therapeutic radioisotopes or hybrid peptides that might offer better therapeutic profiles.The tumor uptake and the tumor/muscle ratio at 1 h p.i. was 1.7 ± 0.3 and 2.1 ± 0.4%ID/g.The highest uptake was observed in kidneys 7.6 ± 0.7%ID/g. [126] [ 177 Lu]Lu-3PRGD 2 0.37, 37.74 and 111 LCC tumor model Biodistribution (at 1, 4, 24 and 72 h p.i.), gamma imaging (at 4 and 24 h p.i.) and maximum tolerated dose (MTD), immunohistochemistry and hematoxylin-eosin staining The tumor uptake at 1 h p.i. was 6.0 ± 0.6%ID/g and remained at 1.2 ± 0.2%ID/g 72 h p.i. Highest uptake was observed in the intestine (5.2 ± 0.5%ID/g) and kidney (4.2 ± 1.1) at 1 h p.i.The MTD was greater than 111 MBq per mouse. [127] [ 90  The tumor uptake of [ 90 Y]Y-RAFT-RGD 1 h p.i. was quick and high (9.0 ± 4.3% ID/g) and remained at 1.8 ± 0.7% ID/g 48 h p.i.The highest kidney uptake was 13.9 ± 3.5%ID/g at 1 h p.i.The toxicity findings were as follows: reduction in leukocyte and platelet counts and higher serum creatinine levels in the treated groups (compared to control).Radiation dosimetry extrapolation to humans: the whole-body effective dose was estimated at 0.11 mSv/MBq.[128] Table 10.Cont.

Formulation Therapy Dose (MBq) Murine Model In Vivo Investigation Main Findings
Ref.
[ 177 Lu]Lu-RAFT-RGD 30-37 U-87 MG glioblastoma SPECT/CT (at 1 and 4 h p.i.), toxicity The tumor uptake at 1 and 4 h p.i. was 3.3 ± 0.5%ID/g and 3.8 ± 0.9%ID/g, and remained at 1.6 ± 0.0%ID/g 48 h p.i.The tumor/muscle ratio was ~10 at 1 h p.i.The highest activity levels (~6%ID/g) were detected in the kidneys and the bladder.Toxicity findings: reduction in leukocyte and platelet counts in treated groups (compared to control).
[ The decay of daughters ( 213 Bi and 221 Fr) was also monitored.All the free 221 Fr and the majority of the 213 Bi decayed at 3 h. [124]

Clinical Studies
Table 11 summarizes some clinical studies performed using radiolabeled RGD-based peptides for cancer imaging and therapy.These trials show promising efficacy in terms of tumor detection, staging, and monitoring treatment responses, highlighting their potential in enhancing the precision of cancer diagnostics and therapy.N/A: not available.

Dual-Targeting Peptides
Achieving optimal single-target tumor imaging and therapy is challenging due to receptor heterogeneity, low binding affinities, and suboptimal in vivo pharmacokinetics.These limitations hinder the generation of high-quality diagnostic images and the effective application of monomeric radiopeptides [82].The solution is the development of heterodimeric peptides (HPs) that link two distinct specific peptide ligands [160].To date, only a few HPs for bispecific EGFR and integrin α V β 3 targeting have been described [132,133,135,161], highlighting the novelty and limited availability of such agents.
Yu et al. [133] designed [ 68 Ga]Ga-NOTA-RGD-Cys-6-Ahx-GE11.Chen et al. [161] subsequently investigated the in vitro and in vivo properties of this radiotracer, comparing it with those of monomeric radiopeptides.The superiority of HPs was noticed in terms of binding affinities and tumor uptake in biodistribution and PET/CT imaging studies, as shown in Figure 4. Specifically, in the PET/CT imaging study at 2 h p.i., the tumor uptake values and tumor/muscle ratios were 3.5 ± 0.6%ID/g and 4.4 ± 1.0 for HPs, 2.4 ± 0.3%ID/g and 2.9 ± 0.7 for [ 68 Ga]Ga-NOTA-GE11, and 2.8 ± 0.5%ID/g and 3.1 ± 0.7 for [ 68 Ga]Ga-NOTA-RGD, respectively.However, in all cases, the liver and kidneys presented high activity levels, highlighting the need for further ligand structure modifications to achieve better pharmacokinetics [161,162].

Dual-Targeting Peptides
Achieving optimal single-target tumor imaging and therapy is challenging due to receptor heterogeneity, low binding affinities, and suboptimal in vivo pharmacokinetics.These limitations hinder the generation of high-quality diagnostic images and the effective application of monomeric radiopeptides [82].The solution is the development of heterodimeric peptides (HPs) that link two distinct specific peptide ligands [160].To date, only a few HPs for bispecific EGFR and integrin αVβ3 targeting have been described [132,133,135,161], highlighting the novelty and limited availability of such agents.
Yu et al. [133] designed [ 68 Ga]Ga-NOTA-RGD-Cys-6-Ahx-GE11.Chen et al. [161] subsequently investigated the in vitro and in vivo properties of this radiotracer, comparing it with those of monomeric radiopeptides.The superiority of HPs was noticed in terms of binding affinities and tumor uptake in biodistribution and PET/CT imaging studies, as shown in Figure 4. Specifically, in the PET/CT imaging study at 2 h p.i., the tumor uptake values and tumor/muscle ratios were 3.5 ± 0.6%ID/g and 4.4 ± 1.0 for HPs, 2.4 ± 0.3%ID/g and 2.9 ± 0.7 for [ 68 Ga]Ga-NOTA-GE11, and 2.8 ± 0.5%ID/g and 3.1 ± 0.7 for [ 68 Ga]Ga-NOTA-RGD, respectively.However, in all cases, the liver and kidneys presented high activity levels, highlighting the need for further ligand structure modifications to achieve be er pharmacokinetics [161,162].Braun et al. [135] refined the radiotracer developed by Chen et al. [161].They replaced the cysteine building block with (NH2-propyl)2Gly-OH to achieve a more uniform structure and used NODA as the chelator.PEG spacers were also incorporated.[ 68 Ga]Ga- Braun et al. [135] refined the radiotracer developed by Chen et al. [161].They replaced the cysteine building block with (NH 2 -propyl) 2 Gly-OH to achieve a more uniform structure and used NODA as the chelator.PEG spacers were also incorporated.[ 68 Ga]Ga-NODA-(PEG 3 -GE11-PEG 3 -c(RGDyK)) and [ 68 Ga]Ga-NODA-(PEG 5 -GE11-PEG 5 -c(RGDfK)) were synthesized.In vitro cell (A431) uptake studies demonstrated favorable integrin α V β 3 -specific receptor affinities for these two bispecific agents.However, they did not exhibit receptor-specific interactions with the EGFR in the in vitro studies.These in vitro findings were corroborated by PET/CT imaging in tumor-bearing mice, which showed that the observed tumor uptake was only mediated by integrin α V β 3 and not by EGFR binding [135].
In the same year, Li et al. [132] synthesized [ 64 Cu]Cu-NOTA-RGD-GE11.The bispecific agent was compared to its monomeric units.[ 64 Cu]Cu-NOTA-RGD-GE11 demonstrated significantly enhanced tumor uptake (4.6 ± 0.2%ID/g) compared to monomeric agents (1.2 ± 0.2%ID/g for [ 64 Cu]Cu-NOTA-RGD and 0.8 ± 0.1%ID/g for [ 64 Cu]Cu-NOTA-GE11) at 2 h p.i. in mice bearing BxPC3 xenografts, as shown in Figure 5.The tumor uptake of the HPs was effectively inhibited in the presence of both non-radioactive c(RGDyK) and GE11, suggesting that both peptides exhibited receptor-specific interactions with their respective targets.These findings underscore the potential of dual-targeting peptides in improving the specificity and effectiveness of cancer therapeutics and diagnostics, paving the way for future clinical applications.NODA-(PEG3-GE11-PEG3-c(RGDyK)) and [ 68 Ga]Ga-NODA-(PEG5-GE11-PEG5c(RGDfK)) were synthesized.In vitro cell (A431) uptake studies demonstrated favorable integrin αVβ3-specific receptor affinities for these two bispecific agents.However, they did not exhibit receptor-specific interactions with the EGFR in the in vitro studies.These in vitro findings were corroborated by PET/CT imaging in tumor-bearing mice, which showed that the observed tumor uptake was only mediated by integrin αVβ3 and not by EGFR binding [135].
In the same year, Li et al. [132] synthesized [ 64 Cu]Cu-NOTA-RGD-GE11.The bispecific agent was compared to its monomeric units.[ 64 Cu]Cu-NOTA-RGD-GE11 demonstrated significantly enhanced tumor uptake (4.6 ± 0.2%ID/g) compared to monomeric agents (1.2 ± 0.2%ID/g for [ 64 Cu]Cu-NOTA-RGD and 0.8 ± 0.1%ID/g for [ 64 Cu]Cu-NOTA-GE11) at 2 h p.i. in mice bearing BxPC3 xenografts, as shown in Figure 5.The tumor uptake of the HPs was effectively inhibited in the presence of both non-radioactive c(RGDyK) and GE11, suggesting that both peptides exhibited receptor-specific interactions with their respective targets.These findings underscore the potential of dual-targeting peptides in improving the specificity and effectiveness of cancer therapeutics and diagnostics, paving the way for future clinical applications.

Conclusions and Future Directions
Herein, we undertook a comprehensive review of the development and application of EGFR-and integrin α V β 3 -targeting peptides as potential radiometal-labeled radiopharmaceuticals for cancer theranostics.The use of these peptides in both diagnostic and therapeutic contexts offers a dual benefit by enabling precise tumor imaging and targeted therapy, potentially leading to better patient outcomes.
Despite the promising aspects of peptide-based radiopharmaceuticals, several challenges and limitations persist.One major challenge is the inherent lower binding affinity and the rapid clearance from the bloodstream of peptides, which can limit their therapeutic efficacy and diagnostic accuracy.Also, the renal toxicity associated with radiometal-labeled peptides accumulation poses a significant concern for patient safety.The small size of peptides, while beneficial for tumor penetration, also contributes to their rapid degradation and clearance, necessitating frequent or higher dosages.
To overcome these challenges, several emerging strategies and technologies are being explored.One approach is the modification of peptides to enhance their stability and binding affinity.This includes the use of cyclization, PEGylation, and the incorporation of non-natural amino acids, which can improve metabolic stability and reduce renal clearance.Another strategy is the development of multivalent and multimeric peptide systems that can increase functional affinity and selectivity towards target receptors.Additionally, the exploration of alternative targeting moieties, such as small-molecule ligands or scaffold proteins, offers a potential avenue for increasing the therapeutic index of these potential radiopharmaceuticals.
The clinical translation of EGFR-and integrin α V β 3 -targeting peptides faces several hurdles that must be addressed in order to realize their full potential.The optimization of peptide structures to enhance receptor binding and stability, coupled with advanced radiolabeling techniques, is crucial for improving the efficacy and safety profiles of these agents.Clinical trials are essential for evaluating the therapeutic benefits, potential side effects, and overall patient outcomes associated with these novel potential radiopharmaceuticals. Furthermore, regulatory approval will be pivotal in determining the feasibility of incorporating these targeted therapies into standard clinical practice.
While EGFR-and integrin α V β 3 -targeting peptides hold significant promise for enhancing cancer diagnosis and treatment, extensive research and development are still required to address the existing challenges.With continued advancements in peptide engineering and radiolabeling technologies, these agents can become integral components of precision oncology, offering more effective and personalized treatment options for cancer patients.In addition, while the targeting of EGFR and integrin is a valid therapeutic strategy, it is the downstream signaling pathways, particularly PI3K/AKT/mTOR and MAPK, that drive cancer progression and therapeutic resistance.Future research and therapeutic development should focus on these pathways to achieve more effective cancer treatments.By integrating inhibitors of these pathways with EGFR-targeted therapies, it may be possible to enhance treatment efficacy and overcome resistance mechanisms, leading to better patient outcomes.

Conflicts of Interest:
The authors declare no conflicts of interest.

Table 8 .
Preclinical studies performed with PET tracers targeting integrin αVβ3.
N/A: not available.